Fecal carriage and clonal dissemination of blaNDM-1 carrying Klebsiella pneumoniae sequence type 147 at an intensive care unit in Lao PDR

Objectives Carbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019. Methods Data regarding clinical conditions, infection control, and antibiotic usage were collected during admission. Rectal swab samples (n = 137) collected during admission were inoculated to selective chromogenic agars, followed by confirmatory tests for extended-spectrum beta-lactamases and carbapenemases. All CPE isolates were sequenced on Illumina (HiSeq2500), reads assembled using SPAdes 3.13, and the draft genomes used to query a database (https://www.genomicepidemiology.org) for resistome, plasmid replicons, and sequence types (ST). Optical DNA mapping (ODM) was used to characterize plasmids and to determine location of resistance genes. Minimum spanning tree was generated using the Bacterial Isolate Genome Sequence database (BIGSdb) and annotated using iTOL. Result From 47 Enterobacterales isolated on selective agars, K. pneumoniae (25/47) and E. coli (12/47) were the most prevalent species, followed by K aerogenes (2/47), K. variicola (1/47), and K. oxytoca (1/47). The overall prevalence of ESBLs was 51.0%; E. coli 83.3% (10/12) and Klebsiella spp. 41.3% (12/29). Twenty percent of the K. pneumoniae (5/25) isolates were carbapenem-resistant, and 4/5 contained the blaNDM-1 gene. All blaNDM-1 isolates belonged to ST147 and were indistinguishable with cgMLST. ODM showed that the blaNDM-1 gene was located on identical plasmids in all isolates. Conclusion The prevalence of ESBL-producing Enterobacterales was high, while carbapenemases were less common. However, the detection of clonal dissemination of blaNDM-1-producing K. pneumoniae isolates in one of the intensive care units calls for vigilance. Stringent infection prevention and antimicrobial stewardship strategies are highly important measures.


Introduction
Antimicrobial resistance is one of the major crises in human and animal health [1]. In a recent systematic analysis of globally available data, antimicrobial resistance was estimated to cause huge loss of economy and life in the coming decades [2]. Resistance to the most clinically important antimicrobial classes, third-generation cephalosporins and carbapenems, is a global problem [3].
Enzymatic hydrolysis of the β-lactam ring of third generation cephalosporins by extended spectrum-β-lactamases (ESBLs) is one of the main mechanisms of resistance. The bla CTX-M-15 gene is widely spread, and is the predominant ESBL-gene encoded by most epidemic isolates [3]. Similarly, in carbapenemase-producing Enterobacterales (CPE) the most important mechanism of resistance for epidemiological purposes is the production of carbapenemases. The bla NDM , bla KPC , bla VIM , bla IPM and bla OXA-48 -group are the most common carbapenemaseencoding genes-mostly located on plasmids [4][5][6][7]. The prevalence, and distribution of carbapenemase-encoding genes varies considerably in different parts of the world. In Southeast Asia, bla NDM has been reported as the most prevalent carbapenemase in India, Thailand, and Vietnam [8,9].
In a previous survey conducted at six hospitals in Lao PDR, the overall antibiotic usage was 71% and varied between hospitals from 50% to 88%. The most commonly prescribed antibiotic was ceftriaxone (40%), and the most common inappropriate use was for surgical prophylaxis (99%) [10]. Meanwhile, the Lao PDR has developed a national action plan for tackling antimicrobial resistance and inappropriate use of antimicrobials. The strategic plan aims to reduce the use of antimicrobials by 20% and healthcare-associated infections by 30%, by 2023 (https:// www.who.int/publications/m/item/lao-pdr-national-strategic-plan-on-antimicrobialresistance-in-lao-pdr-2019-2023). For the strategic plan to meet the set goals, knowledge of local epidemiology is important. Data regarding resistant strains, spread of high-risk clones and mechanism of resistance, including epidemiologically important plasmids, are critical to curb the spread of antimicrobial resistance. Moreover, cost effective and innovative diagnostic technologies that can be used at the site with less technical complexity is equally desired. Generally, it is important to fully understand the magnitude and dynamic of antimicrobial resistance, practice of antibiotic selection, implementation of infection prevention, and antimicrobial stewardship programs in Lao PDR. In this context, the intensive care units are considered hot spots for emergence of antimicrobial resistance and are therefore well suited for sentinel surveillance.
In this study, we determined colonization rate of ESBL-E and CRE among patients admitted to intensive care units of five selected hospitals in Lao PDR. Then, we characterized molecular features of carbapenemase-producing isolates and the plasmids carrying the gene.

Patient characteristics
Patients admitted to intensive care units at five selected hospitals in Laos PDR were enrolled in the study from August to September 2019. The study was ethically approved by the Ministry of Health University of Health Sciences Ethical Committee, Lao PDR (recordal 062-18). A total of 137 rectal swab samples, a single swab from each patient, were collected from all patients admitted to the intensive care units. Clinical and demographic data and were collected by using structured electronic case report forms and entered into Google Forms.

Study site
The study was conducted in five central hospitals in Laos: Mittaphab, Mahosot, Sethathirath, Women and Children's Hospital, and Children Hospital. Mittaphab hospital, specialized in traumatology, has 538 beds, and had an average of 300 bed occupancy per day. Mahosot is a 630 beds hospital with 400 bed occupancy per day. Sethathirath hospital, specialized in endocrines, cancer, and HIV/AIDS, has 250 beds, with full occupancy per day. The women's and children's hospital has 150 beds with full occupancy per day. The children hospital has 70 beds with 100 patients per day (https://data.humdata.org/dataset/who-data-for-lao-people-sdemocratic-republic, https://www.healthdata.org/laos).

Isolation, identification and characterization of bacterial isolates
Rectal swabs were inoculated to chromogenic agar for Enterobacterales (CHROMID 1 ESBL, bioMerieux). Pink (E. coli) and blue (Klebsiella spp., Enterobacter spp) colonies were isolated and species identification was done by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometery at the clinical microbiology laboratory, Karolinska University Hopsital. ESBL-phenotyping was performed primarily by screening using CHROMI-D 1 ESBL agar, followed by combined disk test, a confirmatory test using cefotaxime (Oxoid) and ceftazidime (Oxoid) with and without clavulanic acid (Oxoid).
Antimicrobial susceptibility testing was performed using disk-diffusion method, (https:// www.eucast.org/ast_of_bacteria/disk_diffusion_methodology/). Genomic DNA was extracted using EZ1 1 DNA Tissue Kit (QIAGEN) by using the EZ1 Advanced DNA Bacteria Card on EZ1 Advanced extraction system. NEXTRA-XT kits were used for library preparation and sequencing was performed on Illumina (HiSeq2500) platform at Science for Life Laboratories, Stockholm.

Optical DNA mapping of plasmids
Plasmids were extracted using MACHERY-NAGEL kits, and characterized by optical DNA mapping (ODM) as described previously [11,12]. Briefly, the extracted plamsids were mixed with Cas9 protein (600 ng, Sigma Aldrich) in the presence of equimolar amounts of crRNA (5'CCGCTGCATTGATGCTGAGC3' for the bla NDM-1 gene, and 5' CCGTCGCGATGTAT-TAGCGT 3' for the bla CTX-M-15 gene) and tracrRNA (0.5 nmol each, Dharmacon Inc.) in 1X NEB-3 buffer (New England Biolabs) and 1X bovine serum albumin (BSA, 0.1 μg/ml) and incubated at 4˚C for 30 min. Post Cas9-cutting, the samples were subjected to a one-step competitive binding labeling at 50˚C for 30 min in presence of YOYO-1 (1:2 bp molar ratio, Invitrogen) and Netropsin (60:1 bp molar ratio, Sigma-Aldrich) [13]. λ-DNA (48,502 bp, New England Biolabs) was added as an internal size reference [14]. ODM was performed using nanofluidic devices consisting of 120 parallel nanochannels of dimensions 100 nm (height), 150 nm (width) and 500 μm (length), the fabrication details of the nanofluidic devices is discussed in detail elsewhere [15]. Images were collected using an inverted fluorescence microscope (Zeiss AxioObserver.Z1) with 100X oil objective (Zeiss, 1.46 N.A.), FITC filter (488 nm excitation and 530/50 nm emission) and a Photometrix Evolve sCMOS camera. Image processing was performed using custom written Matlab codes as discussed previously [11,16]. Briefly, kymographs were generated from the images, processed to generate barcodes, intensity patterns along the DNA originating from the variation in YOYO-1: Netropsin binding at GC and AT rich regions, respectively. Barcodes from different experiments were compared against each other to identify any similarity between them. If p < 0.01, the barcodes were considered similar and if p > 0.01, the barcodes were considered not similar.

Ethical approval
Ethical clearance was obtained from Ministry of Health, University of Health Sciences Ethics Comittee, Loa PDR (recordal 062-18). Informed, and written consent was obtained from each study subjects, for minors consent was obtained from either parents or legal guardians.
A majority of the patients (73.7%) had not taken any antibiotic within three months before admission to the hospitals, but when used the most commonly used were oral and interavenous cephalosporins and penicillins (S1
Five (20%) of the K. pneumoniae isolates were resistant to meropenem and four of them which encoded the bla NDM-1 gene were detected at one hospital. All the bla NDM-1 encoding isolates additionally encoded multiple resistance genes, including bla CTXM-15. Furthermore, all the carbapenemase-producing isolates encoded several resistance genes for different classes of antibiotics (aminoglycosides, extended-spectrum cephalosporins, fluoroquinoles, phenicoles, sulphonamides, and trimethoprim-sulfamethoxazole, S1 Table).

Core genome analysis (cgMLST) and plasmid replicon typing
Plasmid replicon typing showed the presence of three plasmids (IncFIB(K), IncFIB(pQil) and IncR) in the bla NDM-1 encoding isolates and these isolates also encoded KL64 capsular type and the O2v1 O-LPS. The type of plasmid replicon, capsular locus, and O-LPS showed that the four carbapenemase-producing strains (L1-L4) encoded similar profiles (S2 Table). A cgMLST analysis showed that they belong to ST147 and that they had identical cgMLST profiles. L5 (ST219) was unrelated to the other four isolates and was not found to encode any carbapenemase genes (Fig 2).

Location of the bla CTX-M-15 and bla NDM-1 on identical plasmids
We have recently demonstrated that plasmid typing using ODM can be a useful method to trace clonal bacterial spread [17]. ODM is a method that in a single experiment yields the number of different plasmids in a sample, their size and on which plasmid a specific (resistance) gene is located, by forming a "barcode" along the DNA [16,18]. From the ST147 strains encoding the bla CTX-15 and bla NDM-1 genes, plasmids were extracted and analyzed by ODM. The barcodes from the ODM experiments showed that the bla NDM-1 gene was present on a

PLOS ONE
plasmid of length around 54 kb in all four isolates. Both the barcodes and gene locations were identical between all the four isolates (Fig 3A, p<0.01). Next, we did the same experiment, but instead targeting the bla CTX-M-15 gene (Fig 3B). For L1, L3 and L4 we observed that the bla CTX-M-15 gene was located on the same plasmid as the bla NDM-1 gene and that the two genes were separated by approximately 10 kb (Fig 3C). However, for L2 the bla CTX-M-15 gene was located on another plasmid with a size of 118 kb.
We further investigated if the 118 kb plasmid was present in any of the other three bla NDM-1 encoding isolates. We identified a similar plasmid of size~110 kb (p < 0.01) in isolate L4, but not in L1 and L3. The 110 kb plasmid in L4 did not encode bla NDM-1 or bla CTX-M-15 . Further, we identified a 174 kb plasmid in isolates L1, L3 and L4 that did not encode bla NDM-1 or bla CTX-M-15 (S4 Fig).

Discussion
Herein, we screened for gut colonization of ESBL-E and CRE among patients admitted to intensive care units at five selected hospitals in Lao PDR in August and September in 2019. A previous systematic review and meta-analysis has shown that patients admitted to the intensive care units are more likely to be colonized with ESBL-producing Enterobacteriaceae, and previous colonization was found to be risk factor of infection [19]. Hence, detection of carbapenem-resistant isolates at an intensive care unit severely compromises safety of patients admitted to the ward. Colonization rates for CRE in neighbouring countries are high [20][21][22], calling for vigilance also in Laos.
The colonization rate detected was 17.5% for ESBL-E and 3.6% for CRE. Carbapenem resistance and the bla NDM-1 genes were detected only in K. pneumoniae. All the CR-KP isolates were found to encode multiple resistance genes, including bla NDM-1 and bla CTX-M-15 . In addition, these isolates were highly similar in all typing methods performed. Most importantly, all the four bla NDM-1 encoding isolates were indistinguishable based on cgMLST phylogenetic analysis, suggesting that there was a small clonal outbreak in the hospital.
Moreover, we characterized plasmids from the CR-KP isolates with ODM, which demonstrated that the bla NDM-1 gene was located at same location on identical plasmids in all isolates. Interestingly, for three out of four isolates, the bla CTXM-15 gene was located on the same plasmid as the bla NDM-1 gene, indicating that the two genes were co-transmitted. The potential use of ODM of plasmids to identify clonal spread of bacteria in hospital settingss has been discussed before in studies at a tertiary hospital in Ethiopia [11], and at a neonatal intensive care unit in Sweden [23]. The results obtained here confirm the potential use of ODM for this purpose. Emergence of multidrug-resistant isolates with the potential to spread through either expansion of the high-risk clone as ST147, or via epidemic plasmids by horizontal genetic transfer, is challenging. The general situation of carbapenemases in Loa PDR is unknown, however, neighboring regions like Thailand, Vietnam, and southern parts China have reported higher prevalence of carbapenemases. Although carbapenems are not yet on the national list of essential drugs in Laos, the high prevalence of ESBL-producing Enterobacterales in Vientiane has driven widespread use of carbapenems imported by individual pharmacies from neighbouring countries. One previous study reported the bla NDM-1 gene from E. coli and A. baumannii [24], and the prevalence carbapenemases might be on the rise despite the lack of sufficient evidence. In 2017, infection prevention, and control strategies were nationally adopted in Lao PDR, and instituted at intensive care units in these hospitals. However, the bla NDM-1 colonization was detected from patients admitted to a special area in the ward, where observation of adherence to infection control measures was routinely performed. These patients were mainly admitted to the hospital for non-communicable diseseases like stroke, hemorrhage, and traumatic brain injury, at the intensive care unit in the same hospital. On the other hand, antibiotic usage was high after admission to the intensive care units. In the presence of high colonization rate of ESBL-E circulating, and the bla NDM-1 gene emerging at these hospitals, strict regulation of prescription, and routine antimicrobial susceptibility testing is important. Moreover, evaluating and strengthening of the current infection prevention and control strategy is necessary.

Conclusions
The emergence of ESBL-E and CRE isolates at an intensive care unit in low-income countries like Lao PDR is concerning. Moreover, clonal dissemination of the high-risk K. pneumoniae clone carrying an epidemic, conjugative plasmid, which in turn encodes mutiple resistance genes is a potential severe burden for a low-income country like Lao PDR. Systematic surveillance, strict prescription policy, and re-defining the infection prevention protocols are important strategies to curb the spread of these strains. Characterization of the epidemic plasmids using ODM can support in strengthening the infection prevention and control strategies in low-income settings.